Huntington's disease (HD), the most common adult onset heritable neurodegenerative disease, is caused by a polyglutamine (polyQ) expansion in the huntingtin (htt) protein and characterized by aggregation of htt into visible intracellular structures called inclusion bodies (IBs). When this project began, the field was divided about the role of IBs in HD; conventional approaches could not resolve the issue. To advance our understanding of HD pathogenesis, we developed a robotic microscope to observe htt induce degeneration and to relate specific molecular changes in each neuron to its ultimate fate. We discovered that levels of diffuse mutant htt predict when neurons degenerate. Remarkably, IB formation led to an abrupt drop in levels of diffuse mutant htt and improved survival compared to similar neurons that did not form IBs. We concluded that IB formation can be a coping response to toxic species of diffuse mutant htt. If IB formation is not the source of HD pathology, what is? Our preliminary data show that some neurons clear IBs altogether and that levels of diffuse mutant htt remain low even after IBs are gone. We hypothesize that IB formation is one part of a broad beneficial adaptive response by neurons to toxic species of malfolded proteins. This response upregulates proteasome- and autophagy-dependent turnover pathways that shorten the half-life of htt, reducing levels of toxic htt species and leading to the clearance of IBs. In Aim 1, we will test this idea by identifying pathways that govern htt metabolism and determining if IB formation is associated with beneficial adaptive changes in htt turnover. We will exploit new technology and our robotic microscope to optically pulse-label htt in individual neurons and follow their fates. In Aim 2, we will determine the extent to which mutant htt induces autophagy in neurons and if the induction of autophagy predicts which neurons will survive. Finally, since htt is required for survival, what htt species is toxic? Does it have a structure that could reveal how it causes degeneration? During the previous period, we made a monoclonal antibody, 3B5H10, that binds a species of diffuse mutant htt and strongly predicts degeneration. To surpass the ability of our robotic microscope to resolve these species, we used crystallography and small-angle x-ray scattering to find that 3B5H10 recognizes a compact structure of polyQ within a monomer of mutant htt. In Aim 3, we will define the structure of the compact conformation of disease-associated polyQ that is recognized by 3B5H10 and use a single-chain version of 3B5H10 expressed in neurons to begin to relate this structure back to the neurodegenerative processes that are the focus of the first two aims. This project is significant because it will elucidate pathogenic mechanisms and therapeutic targets for HD, as it did during the previous period; the findings should also be relevant to Alzheimer's and Parkinson's disease.